Histoplasty Modification of the Tumor Microenvironment in a Murine Preclinical Model of Breast Cancer.

PURPOSE: To develop a noninvasive therapeutic approach able to alter the biophysical organization and physiology of the extracellular matrix (ECM) in breast cancer. MATERIALS AND METHODS: In a 4T1 murine model of breast cancer, histoplasty treatment with a proprietary 700-kHz multielement therapy transducer using a coaxially aligned ultrasound (US) imaging probe was used to target the center of an ex vivo tumor and deliver subablative acoustic energy. Tumor collagen morphology was qualitatively evaluated before and after histoplasty with second harmonic generation. Separately, mice bearing bilateral 4T1 tumors (n = 4; total tumors = 8) were intravenously injected with liposomal doxorubicin. The right flank tumor was histoplasty-treated, and tumors were fluorescently imaged to detect doxorubicin uptake after histoplasty treatment. Next, 4T1 tumor-bearing mice were randomized into 2 treatment groups (sham vs histoplasty, n = 3 per group). Forty-eight hours after sham/histoplasty treatment, tumors were harvested and analyzed using flow cytometry. RESULTS: Histoplasty significantly increased (P = .002) liposomal doxorubicin diffusion into 4T1 tumors compared with untreated tumors (2.12- vs 1.66-fold increase over control). Flow cytometry on histoplasty-treated tumors (n = 3) demonstrated a significant increase in tumor macrophage frequency (42% of CD45 vs 33%; P = .022) and a significant decrease in myeloid-derived suppressive cell frequency (7.1% of CD45 vs 10.3%; P = .044). Histoplasty-treated tumors demonstrated increased CD8+ (5.1% of CD45 vs 3.1%; P = .117) and CD4+ (14.1% of CD45 vs 11.8%; P = .075) T-cell frequency. CONCLUSIONS: Histoplasty is a nonablative focused US approach to noninvasively modify the tumor ECM, increase chemotherapeutic uptake, and alter the tumor immune microenvironment.

Nephronectin is required to maintain right lung lobar separation during embryonic development.

Nephronectin (NPNT) is a basement membrane (BM) protein and high-affinity ligand of integrin α8ß1 that is required for kidney morphogenesis in mice. In the lung, NPNT also localizes to BMs, but its potential role in pulmonary development has not been investigated. Mice with a floxed Npnt allele were used to generate global knockouts (KOs). Staged embryos were obtained by timed matings of heterozygotes and lungs were isolated for analysis. Although primary and secondary lung bud formation was normal in KO embryos, fusion of right lung lobes, primarily the medial and caudal, was first detected at E13.5 and persisted into adulthood. The lung parenchyma of KO mice was indistinguishable from wild-type (WT) and lobe fusion did not alter respiratory mechanics in adult KO mice. Interrogation of an existing single-cell RNA-seq atlas of embryonic and adult mouse lungs identified Npnt transcripts in mesothelial cells at E12.5 and into the early postnatal period, but not in adult lungs. KO embryonic lungs exhibited increased expression of laminin α5 and deposition of collagen IV in the mesothelial BM, accompanied by abnormalities in collagen fibrils in the adjacent stroma. Cranial and accessory lobes extracted from KO embryonic lungs fused ex vivo when cultured in juxtaposition, with the area of fusion showing loss of the mesothelial marker Wilms tumor 1. Because a similar pattern of lobe fusion was previously observed in integrin α8 KO embryos, our results suggest that NPNT signaling through integrin α8, likely in the visceral pleura, maintains right lung lobe separation during embryogenesis.

Estrogen receptor blockade and radiation therapy cooperate to enhance the response of immunologically cold ER+ breast cancer to immunotherapy.

BACKGROUND: Most patients with estrogen receptor positive (ER+) breast cancer do not respond to immune checkpoint inhibition (ICI); the tumor microenvironment (TME) of these cancers is generally immunosuppressive and contains few tumor-infiltrating lymphocytes. Radiation therapy (RT) can increase tumor inflammation and infiltration by lymphocytes but does not improve responses to ICIs in these patients. This may result, in part, from additional effects of RT that suppress anti-tumor immunity, including increased tumor infiltration by myeloid-derived suppressor cells and regulatory T cells. We hypothesized that anti-estrogens, which are a standard of care for ER+ breast cancer, may ameliorate these detrimental effects of RT by reducing the recruitment/ activation of suppressive immune populations in the radiated TME, increasing anti-tumor immunity and responsiveness to ICIs. METHODS: To interrogate the effect of the selective estrogen receptor downregulator, fulvestrant, on the irradiated TME in the absence of confounding growth inhibition by fulvestrant on tumor cells, we used the TC11 murine model of anti-estrogen resistant ER+ breast cancer. Tumors were orthotopically transplanted into immunocompetent syngeneic mice. Once tumors were established, we initiated treatment with fulvestrant or vehicle, followed by external beam RT one week later. We examined the number and activity of tumor infiltrating immune cells using flow cytometry, microscopy, transcript levels, and cytokine profiles. We tested whether fulvestrant improved tumor response and animal survival when added to the combination of RT and ICI. RESULTS: Despite resistance of TC11 tumors to anti-estrogen therapy alone, fulvestrant slowed tumor regrowth following RT, and significantly altered multiple immune populations in the irradiated TME. Fulvestrant reduced the influx of Ly6C+Ly6G+ cells, increased markers of pro-inflammatory myeloid cells and activated T cells, and augmented the ratio of CD8+: FOXP3+ T cells. In contrast to the minimal effects of ICIs when co-treated with either fulvestrant or RT alone, combinatorial treatment with fulvestrant, RT and ICIs significantly reduced tumor growth and prolonged survival. CONCLUSIONS: A combination of RT and fulvestrant can overcome the immunosuppressive TME in a preclinical model of ER+ breast cancer, enhancing the anti-tumor response and increasing the response to ICIs, even when growth of tumor cells is no longer estrogen sensitive.

Effect of matrix heterogeneity on cell mechanosensing.

Cells sense mechanical signals within the extracellular matrix, the most familiar being stiffness, but matrix stiffness cannot be simply described by a single value. Randomness in matrix structure causes stiffness at the scale of a cell to vary by more than an order of magnitude. Additionally, the extracellular matrix contains ducts, blood vessels, and, in cancer or fibrosis, regions with abnormally high stiffness. These different features could alter the stiffness sensed by a cell, but it is unclear whether the change in stiffness is large enough to overcome the noise caused by heterogeneity due to the random fibrous structure. Here we used a combination of experiments and modeling to determine the extent to which matrix heterogeneity disrupts the potential for cell sensing of a locally stiff feature in the matrix. Results showed that, at the scale of a single cell, spatial heterogeneity in local stiffness was larger than the increase in stiffness due to a stiff feature. The heterogeneity was reduced only for large length scales compared to the fiber length. Experiments verified this conclusion, showing spheroids of cells, which were large compared to the average fiber length, spreading preferentially toward stiff inclusions. Hence, the propagation of mechanical cues through the matrix depends on length scale, with single cells being able to sense only the stiffness of the nearby fibers and multicellular structures, such as tumors, also sensing the stiffness of distant matrix features.

Photocleavable Surfactant-Enabled Extracellular Matrix Proteomics.

The extracellular matrix (ECM) provides an architectural meshwork that surrounds and supports cells. The dysregulation of heavily post-translationally modified ECM proteins directly contributes to various diseases. Mass spectrometry (MS)-based proteomics is an ideal tool to identify ECM proteins and characterize their post-translational modifications, but ECM proteomics remains challenging owing to the extremely low solubility of the ECM. Herein, enabled by effective solubilization of ECM proteins using our recently developed photocleavable surfactant, Azo, we have developed a streamlined ECM proteomic strategy that allows fast tissue decellularization, efficient extraction and enrichment of ECM proteins, and rapid digestion prior to reversed-phase liquid chromatography (RPLC)-MS analysis. A total of 173 and 225 unique ECM proteins from mouse mammary tumors have been identified using 1D and 2D RPLC-MS/MS, respectively. Moreover, 87 (from 1DLC-MS/MS) and 229 (from 2DLC-MS/MS) post-translational modifications of ECM proteins, including glycosylation, phosphorylation, and hydroxylation, were identified and localized. This Azo-enabled ECM proteomics strategy will streamline the analysis of ECM proteins and promote the study of ECM biology.

The Mammary Tumor Microenvironment.

A developing breast tumor is regulated by its tumor microenvironment (TME) which includes various immune cell subsets, fibroblasts, adipocytes, and endothelial and epithelial cells surrounded by an extracellular matrix (ECM). Breast tissue density is also a defining feature of breast cancer and plays an integral role in the exchange of biochemical cues between cells and the ECM. Cell signals from these interactions regulate tumor growth, metabolism, immunity, and invasion. The distinct organization of cells in the ECM generates structural patterns that are important in understanding disease development and progression. In this chapter, we discuss this complex interplay between the ECM and cells in the TME. Various models that mimic density are described to more fully understand the effect of ECM density on immunity, metabolism, tumorigenesis, and dormancy. Continued study of the interactions between cells and the ECM in the TME may provide needed biomarkers and therapeutic targets in breast cancer.

A novel bioreactor for combined magnetic resonance spectroscopy and optical imaging of metabolism in 3D cell cultures.

PURPOSE: Fluorescence lifetime imaging microscopy (FLIM) of endogenous fluorescent metabolites permits the measurement of cellular metabolism in cell, tissue and animal models. In parallel, magnetic resonance spectroscopy (MRS) of dynamic nuclear (hyper)polarized (DNP) 13 C-pyruvate enables measurement of metabolism at larger in vivo scales. Presented here are the design and initial application of a bioreactor that connects these 2 metabolic imaging modalities in vitro, using 3D cell cultures. METHODS: The model fitting for FLIM data analysis and the theory behind a model for the diffusion of pyruvate into a collagen gel are detailed. The device is MRI-compatible, including an optical window, a temperature control system and an injection port for the introduction of contrast agents. Three-dimensional printing, computer numerical control machining and laser cutting were used to fabricate custom parts. RESULTS: Performance of the bioreactor is demonstrated for 4 T1 murine breast cancer cells under glucose deprivation. Mean nicotinamide adenine dinucleotide (NADH) fluorescence lifetimes were 10% longer and hyperpolarized 13 C lactate:pyruvate (Lac:Pyr) ratios were 60% lower for glucose-deprived 4 T1 cells compared to 4 T1 cells in normal medium. Looking at the individual components of the NADH fluorescent lifetime, τ1 (free NADH) showed no significant change, while τ2 (bound NADH) showed a significant increase, suggesting that the increase in mean lifetime was due to a change in bound NADH. CONCLUSION: A novel bioreactor that is compatible with, and can exploit the benefits of, both FLIM and 13 C MRS in 3D cell cultures for studies of cell metabolism has been designed and applied.

The Presence of Cyclooxygenase 2, Tumor-Associated Macrophages, and Collagen Alignment as Prognostic Markers for Invasive Breast Carcinoma Patients.

Inflammation, and the organization of collagen in the breast tumor microenvironment, is an important mediator of breast tumor progression. However, a direct link between markers of inflammation, collagen organization, and patient outcome has yet to be established. A tumor microarray of 371 invasive breast carcinoma biopsy specimens was analyzed for expression of inflammatory markers, including cyclooxygenase 2 (COX-2), macrophages, and several collagen features in the tumor nest (TN) or the tumor-associated stroma (TS). The tumor microarray cohort included females, aged 18 to 80 years, with a median follow-up of 8.4 years. High expression of COX-2 (TN), CD68 (TS), and CD163 (TN and TS) predicted worse patient overall survival (OS). This notion was strengthened by the finding from the multivariate analysis that high numbers of CD163+ macrophages in the TS is an independent prognostic factor. Overall collagen deposition was associated with high stromal expression of COX-2 and CD163; however, total collagen deposition was not a predictor for OS. Conversely, local collagen density, alignment and perpendicular alignment to the tumor boundary (tumor-associated collagen signature-3) were predictors of OS. These results suggest that in invasive carcinoma, the localization of inflammatory cells and aligned collagen orientation predict poor patient survival. Additional clinical studies may help validate whether therapy with selective COX-2 inhibitors alters expression of CD68 and CD163 inflammatory markers.

Mechanical signals regulate and activate SNAIL1 protein to control the fibrogenic response of cancer-associated fibroblasts.

Increased deposition of collagen in extracellular matrix (ECM) leads to increased tissue stiffness and occurs in breast tumors. When present, this increases tumor invasion and metastasis. Precisely how this deposition is regulated and maintained in tumors is unclear. Much has been learnt about mechanical signal transduction in cells, but transcriptional responses and the pathophysiological consequences are just becoming appreciated. Here, we show that the SNAIL1 (also known as SNAI1) protein level increases and accumulates in nuclei of breast tumor cells and cancer-associated fibroblasts (CAFs) following exposure to stiff ECM in culture and in vivo SNAIL1 is required for the fibrogenic response of CAFs when exposed to a stiff matrix. ECM stiffness induces ROCK activity, which stabilizes SNAIL1 protein indirectly by increasing intracellular tension, integrin clustering and integrin signaling to ERK2 (also known as MAPK1). Increased ERK2 activity leads to nuclear accumulation of SNAIL1, and, thus, avoidance of cytosolic proteasome degradation. SNAIL1 also influences the level and activity of YAP1 in CAFs exposed to a stiff matrix. This work describes a mechanism whereby increased tumor fibrosis can perpetuate activation of CAFs to sustain tumor fibrosis and promote tumor metastasis through regulation of SNAIL1 protein level and activity.

Neutrophils drive accelerated tumor progression in the collagen-dense mammary tumor microenvironment.

BACKGROUND: High mammographic density has been correlated with a 4-fold to 6-fold increased risk of developing breast cancer, and is associated with increased stromal deposition of extracellular matrix proteins, including collagen I. The molecular and cellular mechanisms responsible for high breast tissue density are not completely understood. METHODS: We previously described accelerated tumor formation and metastases in a transgenic mouse model of collagen-dense mammary tumors (type I collagen-α1 (Col1α1)(tm1Jae) and mouse mammary tumor virus - polyoma virus middle T antigen (MMTV-PyVT)) compared to wild-type mice. Using ELISA cytokine arrays and multi-color flow cytometry analysis, we studied cytokine signals and the non-malignant, immune cells in the collagen-dense tumor microenvironment that may promote accelerated tumor progression and metastasis. RESULTS: Collagen-dense tumors did not show any alteration in immune cell populations at late stages. The cytokine signals in the mammary tumor microenvironment were clearly different between wild-type and collagen-dense tumors. Cytokines associated with neutrophil signaling, such as granulocyte monocyte-colony stimulated factor (GM-CSF), were increased in collagen-dense tumors. Depleting neutrophils with anti-Ly6G (1A8) significantly reduced the number of tumors, and blocked metastasis in over 80 % of mice with collagen-dense tumors, but did not impact tumor growth or metastasis in wild-type mice. CONCLUSION: Our study suggests that tumor progression in a collagen-dense microenvironment is mechanistically different, with pro-tumor neutrophils, compared to a non-dense microenvironment.

Unexpected softening of a fibrous matrix by contracting inclusions.

Cells respond to the stiffness of their surrounding environment, but quantifying the stiffness of a fibrous matrix at the scale of a cell is complicated, due to the effects of nonlinearity and complex force transmission pathways resulting from randomness in fiber density and connections. While it is known that forces produced by individual contractile cells can stiffen the matrix, it remains unclear how simultaneous contraction of multiple cells in a fibrous matrix alters the stiffness at the scale of a cell. Here, we used computational modeling and experiments to quantify the stiffness of a random fibrous matrix embedded with multiple contracting inclusions, which mimicked the contractile forces of a cell. The results showed that when the matrix was free to contract as a result of the forces produced by the inclusions, the matrix softened rather than stiffened, which was surprising given that the contracting inclusions applied tensile forces to the matrix. Using the computational model, we identified that the underlying cause of the softening was that the majority of the fibers were under a local state of axial compression, causing buckling. We verified that this buckling-induced matrix softening was sufficient for cells to sense and respond by altering their morphology and force generation. Our findings reveal that the localized forces induced by cells do not always stiffen the matrix; rather, softening can occur in instances wherein the matrix can contract in response to the cell-generated forces. This study opens up new possibilities to investigate whether cell-induced softening contributes to maintenance of homeostatic conditions or progression of disease. STATEMENT OF SIGNIFICANCE: Mechanical interactions between cells and the surrounding matrix strongly influence cellular functions. Cell-induced forces can alter matrix properties, and much prior literature in this area focused on the influence of individual contracting cells. Cells in tissues are rarely solitary; rather, they are interspersed with neighboring cells throughout the matrix. As a result, the mechanics are complicated, leaving it unclear how the multiple contracting cells affect matrix stiffness. Here, we show that multiple contracting inclusions within a fibrous matrix can cause softening that in turn affects cell sensing and response. Our findings provide new directions to determine impacts of cell-induced softening on maintenance of tissue or progression of disease.

Multi-Modal Investigation of Metabolism in Murine Breast Cancer Cell Lines Using Fluorescence Lifetime Microscopy and Hyperpolarized 13C-Pyruvate Magnetic Resonance Spectroscopy.

Background/Objectives: Despite the role of metabolism in breast cancer metastasis, we still cannot predict which breast tumors will progress to distal metastatic lesions or remain dormant. This work uses metabolic imaging to study breast cancer cell lines (4T1, 4T07, and 67NR) with differing metastatic potential in a 3D collagen gel bioreactor system. Methods: Within the bioreactor, hyperpolarized magnetic resonance spectroscopy (HP-MRS) is used to image lactate/pyruvate ratios, while fluorescence lifetime imaging microscopy (FLIM) of endogenous metabolites measures metabolism at the cellular scale. Results: HP-MRS results showed no lactate peak for 67NR and a comparatively large lactate/pyruvate ratio for both 4T1 and 4T07 cell lines, suggestive of greater pyruvate utilization with greater metastatic potential. Similar patterns were observed using FLIM with significant increases in FAD intensity, redox ratio, and NAD(P)H lifetime. The lactate/pyruvate ratio was strongly correlated to NAD(P)H lifetime, consistent with the role of NADH as an electron donor for the glycolytic pathway, suggestive of an overall upregulation of metabolism (both glycolytic and oxidative), for the 4T07 and 4T1 cell lines compared to the non-metastatic 67NR cell line. Conclusions: These findings support a complementary role for HP-MRS and FLIM enabled by a novel collagen gel bioreactor system to investigate metastatic potential and cancer metabolism.

[64Cu]Cu-PEG-FUD peptide for noninvasive and sensitive detection of murine pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a chronic lung disease resulting in irreversible scarring within the lungs. However, the lack of biomarkers that enable real-time assessment of disease activity remains a challenge in providing efficient clinical decision-making and optimal patient care in IPF. Fibronectin (FN) is highly expressed in fibroblastic foci of the IPF lung where active extracellular matrix (ECM) deposition occurs. Functional upstream domain (FUD) tightly binds the N-terminal 70-kilodalton domain of FN that is crucial for FN assembly. In this study, we first demonstrate the capacity of PEGylated FUD (PEG-FUD) to target FN deposition in human IPF tissue ex vivo. We subsequently radiolabeled PEG-FUD with 64Cu and monitored its spatiotemporal biodistribution via µPET/CT imaging in mice using the bleomycin-induced model of pulmonary injury and fibrosis. We demonstrated [64Cu]Cu-PEG-FUD uptake 3 and 11 days following bleomycin treatment, suggesting that radiolabeled PEG-FUD holds promise as an imaging probe in aiding the assessment of fibrotic lung disease activity.

Bi-directional signaling: extracellular matrix and integrin regulation of breast tumor progression.

Cell transformation and tumor progression involve a common set of acquired capabilities, including increased proliferation, failure of cell death, self-sufficiency in growth, angiogenesis, and tumor cell invasion and metastasis. The stromal environment consists of many cell types and various extracellular matrix (ECM) proteins that support normal tissue maintenance and which have been implicated in tumor progression. Both the chemical and mechanical properties of the ECM have been shown to influence normal and malignant cell behavior. For instance, mesenchymal stem cells differentiate into specific lineages that are dependent on matrix stiffness, while tumor cells undergo changes in cell behavior and gene expression in response to matrix stiffness. ECM remodeling is implicated in tumor progression and can result in increased deposition of stromal ECM, enhanced contraction of ECM fibrils, and altered collagen alignment and ECM stiffness. Tumor cells respond to changes in ECM remodeling through altered intracellular signaling and cell cycle control that lead to enhanced proliferation, loss of normal tissue architecture, and local tumor cell migration and invasion. This review focuses on the bi-directional interplay between the mechanical properties of the ECM and integrin-mediated signal transduction events in an effort to elucidate cell behaviors during tumor progression.

Model of collective detachment in high-grade serous ovarian cancer demonstrates that tumor spheroids produce ECM to support metastatic processes.

High-grade serous ovarian cancer (HGSOC) metastasizes through transcoelomic spread, with both single cells and spheroids of tumor cells observed in patient ascites. These spheroids may form through single cells that detach and aggregate (Sph-SC) or through collective detachment (Sph-CD). We developed an in vitro model to generate and separate Sph-SC from Sph-CD to enable study of Sph-CD in disease progression. In vitro-generated Sph-CD and spheroids isolated from ascites were similar in size (mean diameter 51 vs 55 µm, p > 0.05) and incorporated multiple ECM proteins. Using the in vitro model, nascent protein labeling, and qRT-PCR, we determined that ECM was produced after detachment. As fibronectin plays a key role in many cell adhesion events, we confirmed that inhibiting RGD-based adhesion or fibronectin assembly reduced Sph-CD-mesothelial adhesion strength under shear stress. Our model will enable future studies to determine factors that favor formation of Sph-CD, as well as allow investigators to manipulate Sph-CD to better study their effects on HGSOC progression.

Effect of hyaluronic acid on microscale deformations of collagen gels.

As fibrous collagen is the most abundant protein in mammalian tissues, gels of collagen fibers have been extensively used as an extracellular matrix scaffold to study how cells sense and respond to cues from their microenvironment. Other components of native tissues, such as glycosaminoglycans like hyaluronic acid, can affect cell behavior in part by changing the mechanical properties of the collagen gel. Prior studies have quantified the effects of hyaluronic acid on the mechanical properties of collagen gels in experiments of uniform shear or compression at the macroscale. However, there remains a lack of experimental studies of how hyaluronic acid changes the mechanical properties of collagen gels at the scale of a cell. Here, we studied how addition of hyaluronic acid to gels of collagen fibers affects the local field of displacements in response to contractile loads applied on length scales similar to those of a contracting cell. Using spherical poly(N-isopropylacrylamide) particles, which contract when heated, we induced displacement in gels of collagen and collagen with hyaluronic acid. Displacement fields were quantified using a combination of confocal microscopy and digital image correlation. Results showed that hyaluronic acid suppressed the distance over which displacements propagated, suggesting that it caused the network to become more linear. Additionally, hyaluronic acid had no statistical effect on heterogeneity of the displacement fields, but it did make the gels more elastic by substantially reducing the magnitude of permanent deformations. Lastly, we examined the effect of hyaluronic acid on fiber remodeling due to localized forces and found that hyaluronic acid partially - but not fully - inhibited remodeling. This result is consistent with prior studies suggesting that fiber remodeling is associated with a phase transition resulting from an instability caused by nonlinearity of the collagen gel.

A Label-Free Segmentation Approach for Intravital Imaging of Mammary Tumor Microenvironment.

The ability to visualize complex and dynamic physiological interactions between numerous cell types and the extracellular matrix (ECM) within a live tumor microenvironment is an important step toward understanding mechanisms that regulate tumor progression. While this can be accomplished through current intravital imaging techniques, it remains challenging due to the heterogeneous nature of tissues and the need for spatial context within the experimental observation. To this end, we have developed an intravital imaging workflow that pairs collagen second harmonic generation imaging, endogenous fluorescence from the metabolic co-factor NAD(P)H, and fluorescence lifetime imaging microscopy (FLIM) as a means to non-invasively compartmentalize the tumor microenvironment into basic domains of the tumor nest, the surrounding stroma or ECM, and the vasculature. This non-invasive protocol details the step-by-step process ranging from the acquisition of time-lapse images of mammary tumor models to post-processing analysis and image segmentation. The primary advantage of this workflow is that it exploits metabolic signatures to contextualize the dynamically changing live tumor microenvironment without the use of exogenous fluorescent labels, making it advantageous for human patient-derived xenograft (PDX) models and future clinical use where extrinsic fluorophores are not readily applicable.

Stromal Characteristics and Impact on New Therapies for Metastatic Triple-Negative Breast Cancer.

The heterogenous nature of triple-negative breast cancer (TNBC) is an underlying factor in therapy resistance, metastasis, and overall poor patient outcome. The lack of hormone and growth factor receptors lends to the use of chemotherapy as the first-line treatment for TNBC. However, the failure of chemotherapy demonstrates the need to develop novel immunotherapies, antibody-drug conjugates (ADCs), and other tumor- and stromal-targeted therapeutics for TNBC patients. The potential for stromal-targeted therapy is driven by studies indicating that the interactions between tumor cells and the stromal extracellular matrix (ECM) activate mechanisms of therapy resistance. Here, we will review recent outcomes from clinical trials targeting metastatic TNBC with immunotherapies aimed at programed death ligand-receptor interactions, and ADCs specifically linked to trophoblast cell surface antigen 2 (Trop-2). We will discuss how biophysical and biochemical cues from the ECM regulate the pathophysiology of tumor and stromal cells toward a pro-tumor immune environment, therapy resistance, and poor TNBC patient outcome. Moreover, we will highlight how ECM-mediated resistance is motivating the development of new stromal-targeted therapeutics with potential to improve therapy for this disease.

Multimodal imaging demonstrates enhanced tumor exposure of PEGylated FUD peptide in breast cancer.

In breast cancer, the extracellular matrix (ECM) undergoes remodeling and changes the tumor microenvironment to support tumor progression and metastasis. Fibronectin (FN) assembly is an important step in the regulation of the tumor microenvironment since the FN matrix precedes the deposition of various other ECM proteins, controls immune cell infiltration, and serves as a reservoir for cytokines and growth factors. Therefore, FN is an attractive target for breast cancer therapy and imaging. Functional Upstream Domain (FUD) is a 6-kDa peptide targeting the N-terminal 70-kDa domain of FN, which is critical for fibrillogenesis. FUD has previously been shown to function as an anti-fibrotic peptide both in vitro and in vivo. In this work, we conjugated the FUD peptide with 20-kDa of PEG (PEG-FUD) and demonstrated its improved tumor exposure compared to non-PEGylated FUD in a murine breast cancer model via multiple imaging modalities. Importantly, PEG-FUD peptide retained a nanomolar binding affinity for FN and maintained in vitro plasma stability for up to 48 h. Cy5-labeled PEG-FUD bound to exogenous or endogenous FN assembled by fibroblasts. The in vivo fluorescence imaging with Cy5-labeled FUD and FUD conjugates demonstrated that PEGylation of the FUD peptide enhanced blood exposure after subcutaneous (SC) injection and significantly increased accumulation of FUD peptide in 4T1 mammary tumors. Intravital microscopy confirmed that Cy5-labeled PEG-FUD deposited mostly in the extravascular region of the tumor microenvironment after SC administration. Lastly, positron emission tomography/computed tomography imaging showed that 64Cu-labeled PEG-FUD preferentially accumulated in the 4T1 tumors with improved tumor uptake compared to 64Cu-labeled FUD (48 h: 1.35 ± 0.05 vs. 0.59 ± 0.03 %IA/g, P < 0.001) when injected intravenously (IV). The results indicate that PEG-FUD targets 4T1 breast cancer with enhanced tumor retention compared to non-PEGylated FUD, and biodistribution profiles of PEG-FUD after SC and IV injection may guide the optimization of PEG-FUD as a therapeutic and/or imaging agent for use in vivo.